1. Field
This document relates generally to cellular measurements based on mid-infrared absorption measurements and particularly, but not by way of limitation, cellular measurements based on mid-infrared absorption measurements using mid-infrared laser based architectures for infrared activated cell sorting (IRACS).
2. Description of the Related Art
Identification, classification and sorting of cells, in particular live cells, is a subject of considerable research and commercial interest. Most recently systems for sorting stem cells have been an area of particular focus. For example, methods for separating cancerous from non-cancerous cells have been demonstrated. For another example, there is an established market for cell sorting for gender offspring selection by identification and selection of X- or Y-bearing spermatozoa.
There is currently no safe and accurate method for cell sorting. The most advanced technology uses fluorescence-activated cell sorting (FACS), where living cells are incubated in a fluorescent DNA-attaching dye, exposed to a high-intensity, high-energy UV laser beam, and sorted according to observed fluorescence. There are two major disadvantages to this method applied to certain cells, including low accuracy and safety concerns. For example, in sperm cell sorting, the FACS process is able to achieve 88% X-enrichment and only 72% Y-enrichment, even at very low sort rates (20-30 per second output). High scattering at UV and visible wavelengths is a major factor. In addition, in sperm cell sorting, the FACS process has been shown to cause chromosomal damage in sperm cells as a result of the dyes used, and as a result of exposure to high intensity 355 nm laser light.
The use of optical methods to identify and classify cells has many potential advantages such as speed, selectivity/specificity, and their non-invasive nature. As a result, a number of methods have been demonstrated in which light is used to interrogate cells and determine critical information. One such method is the use of fluorescent markers, which are chemicals that bind to specific structures or compounds within the target cells and are introduced into the mixture of cells. The mixture is subsequently rinsed to remove excess fluorescent markers and the cells are exposed to intense UV or other short-wavelength radiation in order to “read out” relevant quantities and classify the cell. The chemical markers provide good specificity. However, these chemical markers may damage or alter the function of the target cells, which is particularly disadvantageous for live cell sorting. In testing, dyes used as markers for DNA, for example, have resulted in chromosomal damage. Further, the intense UV or visible light used to read the level of marker in the cell may damage the cell, in particular, DNA damage results from exposure to high-energy UV or visible photons. Also, because of the wavelengths used in so called fluorescence activated cell sorting (FACS) systems, quantitative measurements (rather than yes/no measurements for a particular antibody) are made very difficult, because both the illuminating wavelength and the emitted fluorescence are scattered and absorbed by cellular components. This means that cell orientation becomes an important factor in accurate measurement, and can dramatically reduce the effectiveness of the system. For example, sperm cell sorts for X- and Y-carrying sperm, which measure the differential in DNA between cells, require very specific orientation (only 10% of cells typically meet the orientation criteria), and still provide accuracy only in the 70-90% range for humans.
Another method to interrogate cells and determine critical information is Raman spectroscopy. In Raman Spectroscopy, cells are exposed to intense visible or near infrared (NIR) light. This light is absorbed as a result of molecular bond vibrations within the cellular structure. Secondary emission of photons at slightly different wavelengths occurs, according to Stokes and anti-Stokes energy shifts. Measurement of these wavelengths allows the chemical composition of the cell to be measured. With Raman spectroscopy, the individual photon energy is generally lower than that used for fluorescent markers, however, the net energy absorbed can be very high and unsafe for live cells. Raman scattering is an extremely weak process: typically only 1 in 10^ 10 incident photons give rise to a Raman-shifted photon, thus requiring long exposure times to generate sufficient shifted protons for accurate measurement. While Raman may not be suitable for high-volume live cell sorting, it can be used in conjunction with other methodologies described herein. Higher sensitivity methods such as coherent anti-Stokes Raman scattering (CARS) are being developed which may enable high-throughput screening.
One significant drawback of mid-IR spectroscopy is the strong absorption by water over much of the “chemical fingerprint” range. This has strongly limited the application of Fourier Transform Infrared Spectroscopy (FTIR) techniques to applications involving liquid (and therefore most live cell applications) where long integration times are allowable—so sufficient light may be gathered to increase signal-to-noise ratio and therefore the accuracy of the measurement. The lack of availability of high-intensity, low etendue sources limit the combination of optical path lengths and short integration times that may be applied. In addition, because of the extended nature of the traditional sources used in FTIR, sampling small areas (on the order of the size of a single cell) using apertures further decimates the amount of optical power available to the system.
One approach to enabling liquid or solid-state measurements in the mid-IR is to use surface techniques. A popular method is the use of an attenuated total reflection (ATR) prism that is positioned directly in contact with the substance of interest (sometimes using high pressure in the case of solid samples). Mid-IR light penetrates from the prism up to several microns into the sample, and attenuates the internal reflection according to its wavelength-dependent absorption characteristics.
Another method which was more recently developed is the use of plasmonic surfaces which typically consist of conductive layers patterned to produce resonances at specific wavelengths; at these resonances, there is coupling into substances places on top of the layer, and again, absorption at a specific wavelength may be measured with good signal. Again, however, the coupling into the substance of interest is very shallow, typically restricted to microns.
Analysis of particles including biological cells for size, shape and chemical or biochemical content is of great interest in many applications including medicine, drug discovery, materials science and manufacturing, process control, food and water safety, and other markets. The characterization of particles by optical scattering characteristics is already widely used in such applications. For example, blood counts are performed using scattering-based cytometers that effectively categorize cells according to size, shape and density. For measurement of biochemical content, however, other methods must be used, or combined with scattering techniques. Most commonly, fluorescent dyes or labels are added to achieve this. This adds significant complexity to the measurement process, and limits the applications in which particle size, shape, density and biochemical makeup may be characterized accurately.
One well-known method for assessing biochemical content of condensed phase materials is infrared spectroscopy, usually through the use of a Fourier Transform Infrared (FTIR) spectrometer. In FTIR, absorption spectra of the material under inspection is measured; in the mid-IR range, molecules have specific absorption bands or “fingerprints” corresponding to molecular bond vibrations. These fingerprints may be used to calculate makeup of a sample, chemical concentrations, and even molecular conformations (packing, folding, and other inter- or intra-molecular interactions that are reflected in the bond force/length and therefore its characteristic resonant frequency).
One of the problems raised in mid-IR microspectroscopy when particles are present is that of scattering. First, there is general wavelength dependence in scattering, with scattering cross-section growing as the wavelength becomes shorter compared to the particle(s) being measured. Second, where particle (or medium) components have strong absorption features, there is necessarily also (by the Kramers-Kronig relationship) a resonant feature in the real refractive index of the particle or medium. Since scattering is dependent on both the size of the particle and the refractive index of the particle relative to the medium, this results in localized “resonant” scattering. Many groups have developed algorithms to correct for both the non-resonant and resonant Mie scattering effects in FTIR measurements; most of them based on iterative models that fit an observed IR absorption spectrum.
Mie scattering is dominant when the particles in the path are on the order of the interrogating wavelength. The magnitude and angle of scattering is determined by the size, shape and index of particles relative to the medium. Problems are especially prevalent when the particles or cells being measured have high-index relative to the medium when using mid-IR spectroscopy such as FTIR where scattered light can be misinterpreted as absorption, and artifacts in the Fourier-inverted spectrum can result. Some of the causes or promoters of this scattering loss include: 1) Measurement of cells in air medium, rather than in a water medium. This causes additional index mismatch between the medium and cells, dramatically raising scattering efficiency and angles; 2) Measurement of absorption peaks at high wavenumbers (short wavelengths) where scattering efficiency is higher; 3) Insufficient capture angle on the instrument, where typically the capture angle on these instruments is identical to the input angle, not allowing for light scattered outside of the delivered IR beam angle; and 4) Transflection or other surface-based measurements. These configurations may lead to additional artifacts in conjunction with Mie scattering effects.
In cytometry techniques, visible or near-infrared wavelengths are typically used; by measuring the intensity of scattering over a range of angles the cell size may be estimated. For example, some modern blood count equipment uses this method to approximate blood cell size and shape to generate a detailed blood count. However, the scattering distribution resulting from laser illumination at these wavelengths is dependent on many factors, including cell shape, orientation, density, and chemical composition. It is not possible to determine chemical composition at these wavelengths, and therefore, to eliminate this factor which affects scattering pattern and therefore volume estimate.
The ability to measure particles or cells suspended in liquid, either individually or in aggregate, using mid-IR spectroscopic methods has significant implications in a number of applications, both in the biomedical market and in other markets. Ideally, an optical method could be devised that would estimate the volume of the particle that was chemically distinct from the medium surrounding (and in some cases permeating) it. However, one of the challenges of mid-IR spectroscopy on particles or cells, particularly where high throughput is required, is getting sufficient contrast as a particle passes through the measurement volume. This is particularly acute when multiple wavelengths are used simultaneously (for example, modulated at different carrier frequencies), and each is only absorbed or scattered in a small fraction by the particle(s). For cells suspended in water, the significant absorption bands associated with water in the mid-IR pose a challenge.
In many applications where small particles are measured, it is useful to measure the volume of the particles. A related and often more important measurement is the content in the particle excluding its medium. For example, when measuring biological cells, the non-water volume of a cell can be a strong marker for cell phenotype, and may in addition contain significant information on the status of the cell (for example, if it is actively dividing). Multiple methods for estimating cell volume have been devised.
One device, which approximates volume, is a Coulter Counter, which uses a voltage potential over a channel filled with conductive medium though which biological cells flow; as the cells pass through the channel, they block electrical current, with the reduction in current indicative of cell volume. This device may be used, for example, to differentiate red from white blood cells and rapidly generate a blood count. Coulter Counters are used outside of biology as well in applications such as paint, ceramics, glass and food manufacturing where particle sizing (and distribution of sizing) is of high importance.
This method, while very useful for measuring particle volume, is dependent on the precise composition of the particle or cell, including whether its membrane is electrically insulating and on the conductivity of liquid contained inside the cell. For estimating total non-water (or more generally non-medium) volume, it would be preferable to eliminate this dependency. Additionally, the requirement for an electrically conductive medium places limits on the materials and particles that may be measured with a resistance-based method such as the Coulter Counter. While capacitive methods have been employed as well, these are highly sensitive to particle position and other environmental factors.
In the quest to provide accurate measurements of true weight of a particle, or more specifically a biological cell, one group (Manalis et al at MIT) have gone so far as to build an ultra-sensitive “scale” based on a microfluidic channel on a microfabricated cantilever, though which biological cells are flowed. The characteristic resonant frequency of the cantilever is shifted as each cell passes through the tip of the cantilever, allowing measurement of cell mass. This device has been proposed as a method to repetitively measure individual cell masses through the course of a treatment (for example, as a drug or other treatment is applied to a population of cancer cells). While this method is novel and potentially highly accurate, it is highly complex (requires significant difficult fabrication, calibration, compensation) and potentially suffers from low throughput (flow rates must be kept low to provide an accurate measurement and prevent rapid clogging).
Both of the aforementioned methods (Coulter Counter and cantilever “scale”) also have the disadvantage that they may be difficult to integrate with other measurement techniques. Specifically, in biomedical applications where biological cells are measured, much additional cellular characterization is done optically, by measurement of scattered light and/or fluorescence induced in the cell or chemical dyes/labels that have been added to stain or mark the cell. Ideally, a method for measuring non-medium volume of a particle or cell could be integrated seamlessly with these other measurements to provide an integrated measurement. In other words, an optical method for measuring non-medium volume would be strongly preferred. This would additionally not require a medium that is electrically conductive.
Thus there remains a need for techniques to identify and measure particles or cells that provide accurate results and are usable on living cells.